1. Field of the Invention
The present invention relates to a method for producing a secreted protein in mammalian adherent cell culture.
2. Description of the Related Art
The majority of industrial production processes that utilize Chinese hamster ovary (CHO) host cells use cell lines adapted to grow in suspension, e.g., t-PA (Werner et al., 1993 and Lubiniecki et al., 1994), for which serum-free medium have been available for the last 10 years (Gorfien et al., 1991). However, an alternative strategy for CHO cell culture is to support attached cell growth where cells adhere to a matrix, e.g., a microcarrier (Kadouri, 1994). CHO cells can grow successfully on such supports, and industrial processes have been developed utilizing CHO microcarrier cultures, e.g., Puregonxc3xa2 (Olijve et al., 1996).
The adherent growth of CHO cells on microcarriers is usually accomplished by supplying serum to the culture, in the form of fetal bovine serum (FBS) (Xiao et al., 1994; Clark et al., 1981; Nikolai et al., 1992; Asselbergs et al., 1992; Levin et al., 1992; Watson et al., 1994 and Ohlson et al., 1994). This complex culture medium supplement contains the necessary attachment and growth factors required to promote the adherent phenotype, e.g., fibronectin, laminin (Zaworski et al., 1993 and Nilsson, 1989). Without the presence of serum, it has been reported that some cell lines will not grow on microcarriers (Schmid et al., 1992), or that the absence of serum is detrimental to product formation (Teige et al., 1994). Although serum-free adherent growth of CHO cells has been accomplished using a basal medium supplemented with insulin, transferrin and selenium (Gasser et al., 1985), this growth was obtained in the very passive environment of a T-flask, an environment that is known to differ significantly from the environment encountered in attached growth on microcarriers in a bioreactor (Cherry et al., 1985).
For microcarrier-based industrial processes, cells are usually fed (via continuous perfusion) culture medium supplemented with serum until the cells fill the available surface area provided by the microcarrier (the growth phase). The perfused medium is then switched to a different medium, usually one that is a serum-free formulation, as cells at this point are not dividing and can rely on the existing extracellular matrix to support their attachment. It is during this second phase (the production phase) that conditioned medium is collected for recovery and purification of the recombinant protein. However, in such a two-phase system, the switch from growth to production can lead to a dramatic cellular response, such as cell loss and decreased volumetric productivity of recombinant protein (Cosgrove et al., 1995). Alternatively, a lag phase can occur when the cells respond to the dramatic switch in environment, which then results in a period of low recombinant protein productivity until the cells are able to recover.
Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.
It is an object of the present invention to avoid the problems and difficulties encountered in the prior art as discussed above.
The present invention provides a method for producing a heterologous secreted protein from transformed CHO cells grown on microcarriers in a serum-free cell culture medium. This method has the advantage of overcoming the problem of a production lag phase observed when switching from a serum-containing to a serum-free environment.